Presently, rapid, handheld, or portable instrumentation for determining the quality of fluids, such as natural waters, recreational waters, distributed and treated water supplies, buffers, seawater, milk, biological fluids, and gases, including air does not exist. Conventional analytical techniques, such as mass spectrometry and its variants, are currently being modified for detection and identification of microorganisms. [Holland et al., 1999; Winkler et al., 1999]. Mass spectrometry, which requires various types of sample pretreatment and/or separation, requires that large libraries of chemical signatures be generated in advance of analysis. Also, bacterial mass spectra are very irreproducible even between samples taken from the same strain. Recently, the effects of cell aging on the reproducibility of the mass spectra have been identified as a problem [Arnold et al., 1999]. Equally significant is that the instrumentation is still cumbersome and expensive, requires substantial operator expertise, and requires a large spectra reference library, making this approach unsuitable if the threat is unknown.
Polymerase chain reactors (PCR) are also being developed for detecting microorganisms, and have been recently demonstrated in a portable, if not lightweight, package [Belgrade et al., 1999]. Although very promising, the laboratory and real-time PCR-based instruments are still limited by high cost, user expertise requirements, and analysis time. Micro-machined “chip” devices based on PCR, GC/MS, and flow cytometry are still laboratory curiosities. Portable Surface-enhanced Raman spectrometers are under development for detection of biological warfare agents, but they do not offer the sensitivity or specificity required for rapid detection of pathogens.
Biosensors typically incorporate a molecular receptor such as an antibody or nucleic acid fragment immobilized on a solid substrate. The transduction mechanism can be electrical, electrochemical, optical, or mass-based [Paddle, 1996]. Very elegant work has been done using antibodies and, more recently, DNA probes for detection of pathogenic microorganisms in water [Cao et al., 1995; Wadkins et al., 1998; Stahl, 1999]. Specifically in the area of food borne pathogens, immunoelectrochemical and surface-enhanced infrared sensors have been demonstrated [Brewster et al., 1996; Brown et al., 1998]. These assays, however, are inherently complex, requiring sample preparation, addition of solvents, and washing steps. Even when these steps are automated, they require the development and maintenance of fluid handling systems and reagent reservoirs, and they generate chemical waste. Additionally, producing high quality antibodies is a costly, time-consuming process, and both approaches are subject to non-specific binding. For example, several bacteria of the Escherichia and Salmonella urbana genus have been reported to cross react with antibody raised for E. coli O157 [Goodridge et al, 1999].
The use of aptamer technology has also been proposed for detection of pathogenic microorganisms. Aptamers are nucleic acids that have diverse functionality such as specific ligand-binding characteristics [Potyrailo et al., 1998; German et al., 1998]. Aptamers are generally synthesized using combinatorial chemistry techniques to generate very large, complex libraries of molecules (>1015), which then must be screened, isolated, and amplified for the aptamer of interest. Hennes and Settle have demonstrated that individual viral particles (i.e., bacteriophage) can be ligated with fluorescent reporters with subsequent detection enumeration, and discrimination in a complex biological sample [Hennes et al., 1995a; Hennes et al., 1995b]. Goodridge et al. have demonstrated a similar fluorescent-bacteriophage assay in bulk solution for detecting Escherichia coli O157:H7 [Goodridge et al., 1999]. Viruses stained in this manner were found to adsorb to host cells with high specificity. Moreover, cells with attached fluorescently labeled viruses were clearly distinguishable from non-host cells.